Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase

Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII–(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII–tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues “on the fly” to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

. The sequence of the pET22b_LsAA9 wild-type construct for periplasmic expression (the two key residues W64 and Y164 is highlighted in bold magenta, the pelB sequence is highlighted in blue and the C-terminal twin strep tag is highlighted in orange and green). The associated codon optimized DNA sequence is given below.
To generate copper-loaded LsAA9 WT, W64F, H147F, H147Q, H147A and Q162A, a solution of CuCl 2 (1.1 equiv., 10 mM in Milli-Q water) was slowly added to the above concentrated apo-protein solution followed by incubation on ice for 2h. Excess free copper was removed using a 10DG desalting column with potassium phosphate (50 mM, pH 6.0) as the elution buffer. The Y164F mutant was found to have a weaker affinity for the catalytic copper ion when compared to the wild-type enzyme. It was observed that use of the 10DG desalting column resulted in low concentrations of copper-loaded Y164F. Therefore, to produce the copperloaded Y164F, the concentrated apo-protein solution was incubated with CuCl 2 (0.75 equiv., 10 mM in Milli-Q water) on ice for at least 2 h without any further desalting and concentration steps. The purified proteins were analyzed by SDS-PAGE ( Figure S4c), high-resolution mass spectrometry (HRMS) and electron paramagnetic resonance (EPR) spectroscopy, and stored at 4 ˚C prior to use within one week.

Mass spectrometry (MS) analysis
Purified proteins were buffer exchanged into 0.1% acetic acid using a 10 kDa MWCO Vivaspin (Sartorius) and diluted to a final concentration of 0.5 mg/mL. MS was performed using a 1200 series Agilent LC, 5 μL injection into 5% acetonitrile (with 0.1% formic acid), and desalted inline for 1 min. Protein was eluted over 1 min using 95% acetonitrile with 5% water. The resulting multiply charged spectrum was analysed using an Agilent QTOF 6510 and deconvoluted using Agilent MassHunter Software. The measured protein MS are listed in Table  S2.

Stopped-flow kinetics
Stopped-flow kinetic measurements were carried out using an SX20 rapid mixing stopped-flow spectrophotometer (Applied Photophysics Ltd, Leatherhead, UK) placed inside a Belle Technology anaerobic chamber (oxygen levels < 2 ppm) as previously described. 2 Multiple wavelength data were collected at 3 ˚C using a photodiode array (PDA) detector and single wavelength data was obtained from a photomultiplier tube (PMT) single wavelength detector. Most stopped-flow single mixing experiments were peformed at 3 ˚C in potassium phosphate buffer (KPi 50 mM, pH 6.0, degassed overnight before use) using a final concentration of 50 μM protein (reduced by 25 µM ascobate) and 50 μM oxidants (m-CPBA, peracetic acid and H 2 O 2 ), or H 2 O 2 (500 µM, 2500 µM). For double mixing stopped-flow measurments, reduced Cu I -LsAA9 WT (200 µM, 50 µM final) was first mixed with PAA (1 equiv., 200 µM, 50 µM final) and held in an ageing loop for 50 ms to generate Int1, or 1 s to generate Int2, before mixing with either buffer (as a control) or G5 substrate (50 µM, 100 µM, 200 µM and 500 µM final concentrations) at pH 6.0, 3 ˚C. Stopped-flow fluorescence measurements using a FRET-G4 substrate were performed using an excitation wavelength of 330 nm and a 455 nm highpass filter. Stopped-flow samples were freshly prepared by bringing concentrated enzyme stocks, reductant stocks, oxidants stocks, and cellopentaose (G5) substrate stocks inside the N 2 glovebox.

Stopped-flow kinetics fitting
Raw UV/visible absorbance changes from stopped-flow single mixing experiments were fitted globally using the Pro-Kineticist software (Applied Photophysics). The multi-wavelength stopped-flow data for LsAA9 wild type and variants from a range of different experimental conditions were fit to a sequential kinetic model to extract component spectra and global rate constants. The number of phases was determined by visual inspection of the spectra and global residuals. The fit of the model to selected wavelengths is shown in the supporting information in each case. Kinetic transients at single wavelengths from stopped-flow experiments were fitted to single, double or triple exponential equations using the Pro-Data Viewer software.

Quench-flow kinetics
Quench-flow experiments were carried out using an RQF-73 rapid quench-flow instrument (TgK Scientific, UK) placed inside a Belle Technology anaerobic chamber (oxygen levels < 2 ppm). All quench-flow double mixing experiments were performed at 3˚C in KPi (50 mM, pH 6.0, degassed overnight before use). The reduced Cu I -LsAA9 WT (200 µM, 50 µM final) was first mixed with m-CPBA (1 equiv., 200 µM, 50 µM final) and held in an ageing loop for 50 ms to generate Int1, or 1 s to generate Int2, before mixing with G5 substrate (5 equiv., 250 µM final concentration) at pH 6.0 at 3˚C. The final reaction mixture (200 µL) was recovered from the instrument and immediately quenched with MeCN (200 µL). The mixture was centrifuged at 10 000 rpm for 10 min and the supernatant was subjected to LC-MS analysis.

Liquid Chromatography-Mass Spectrometry
All analysis was conducted on a QExactive Plus with an Ultimate 3000 UHPLC (Thermo, UK). The UHPLC was equipped with a ZIC-cHILIC column (2.1 mm x 100 mm; 3 mm particle size). The solvents employed were (A) water + 0.1% formic acid and (B) acetonitrile + 0.1 % formic acid. The flow gradient was programmed to equilibrate at 99% B for 2 min followed by a linear gradient to 10% B over 10 min and held at 10% B for 2 min before returning to 99% B for 2 min with a flow rate of 600 mL/min. The column was maintained at 40 °C and the samples chilled in the autosampler at 10 °C. A sample volume of 5 μL was injected onto the column. Data acquisition was conducted in full scan mode in the scan range of 90-1050 m/z with a resolution of 70,000, an AGC target of 3e 6 and a maximum integration time of 200 ms. The acquisition was conducted in positive ion mode.

Electron paramagnetic resonance (EPR)
EPR measurements were carried out using a Bruker ELEXSYS-E580 X-band EPR spectrometer capable of operating both in continuous wave (CW) and pulsed modes, equipped with an Oxford variable-temperature unit and ESR900 cryostat with Super High-Q resonator. All EPR samples were prepared in quartz capillary tubes (outer diameter; 4.0 mm, inner diameter 3.0 mm) and frozen immediately in liquid nitrogen until further analysis. The X-band EPR tubes were then transferred into the EPR probe-head, which was pre-cooled to 20 K. A microwave power of 30 dB (0.2 mW) and modulation of 5 G appear to be optimal for recording the EPR spectra of the LsAA9 and its variants. The low temperature EPR spectra were acquired using the following conditions: sweep time of 84 s, microwave power of 0.2 mW, time constant of 81 ms, average microwave frequency of 9.386 GHz and modulation amplitude of 5 G. The intermediates were formed using 200 µM protein with 100 µM ascorbate and 200 µM PAA in 50 mM KPi buffer (pH 6.0). Samples were prepared using a home-made hand mixing device with two syringes, one containing reduced protein and the other containing PAA, that are directly mixed into an EPR tube and quickly frozen in liquid nitrogen. Analysis and simulations of the CW-EPR spectra were performed using EasySpin toolbox (5.2.28) for the /Matlab (R2017a) program package. 3 8 truncated by methyl substitution of the C γ carbon. For the histidyl radical calculation no hydrogen atom was added to the C2 position of His-1. Geometry optimizations were performed with the BP86 12 functional (with RI approximation), Def2-TZVP basis set 13 on Cu and ligating atoms and Def-2-SVP on all the remaining atoms; empirical dispersion correction were accounted using Grimme's D3 method with Becke-Johnson damping (D3BJ) 14 ; solvation effects were included with the conductor-like polarizable continuum model (CPCM, ε=4.0) 15 . The broken symmetry (BS) approach was used to optimize the singlet spin state geometry. Single point energies were calculated using the B3LYP functional 16 and the Def2-TZVP basis set on all atoms. Corrected singlet state energies and exchange coupling constants (J) were computed with the Yamaguchi formula 17 : = -< 2 > -< 2 > Cu K-edge absorption spectra were calculated with time dependent density functional theory (TD-DFT) using ORCA 4.2 software approach applying the Tamm−Dancoff approximation. 18 The XAS absorption spectra were computed with RIJCOSX 19 approximation with a dense integration grid, using the uB3LYP functional and ZORA-def2-TZVP basis set 20 on all atoms. The Zeroth order relativistic approximation (ZORA) was employed to better account for relativistic effects; deemed important for this type of calculation. 21 The SARCJ auxiliary basis set was also used to give improved accuracy on relativistic calculations than that of def2J. The integration grid size was increased to its maximum of 7. Solvation effects were accounted for using the Conductor-like Polarizable Continuum Model (CPCM), ε = 80.0 and refractive index = 1.33. 30 transitions (roots) were calculated starting from the Cu 1s orbitals. The lowest energy peak calculated in both models corresponds to a weak quadrupole allowed 1s-3d transition (final state occupying the Cu hole), the next is a weak 1s→2p MLCT (final state occupying a 2p orbital on the C2 carbon atom of His-1), followed finally by the rising edge of 1s→4p/continuum.            an electron spin S = ½ were considered, Cu II and a radical signal (Table S4). The relative population of the radical signal is < 2% compared to the Cu II signal with the fastest freezequenching time of ~ 1 s. The population of the radical signal goes down when the freezequenching time is slowed down to ~2-5 s, without significant changes in the spin-Hamiltonian parameters of the Cu II and the radical signals.        individual spin-Hamiltonian parameters of the Cu II ion and radical signals were used (see Table   S4 for more details) along with the near rhombic dipolar interaction tensor, T = [+3417 +917 -4334], and J = -100 MHz. It is noteworthy to mention that the magnitude of the exchange interaction is not sensitive to the simulated spectrum, so range of J is plausible. Also, it is noted that T is not axial (usually the case), which is not surprising given the large anisotropy associated with the Cu II ion and the delocalisation of the electron density that often occurs within the tyrosyl radical.  The spin-Hamiltonian parameters used to model the EPR spectrum are given in the Table S4.